Well plate

ABSTRACT

The present invention includes an apparatus for preparing samples for measurement by x-ray fluorescence spectrometry. The apparatus comprises a plate having one or more holes passing through the plate. The holes are covered by a film on one side of the plate. The holes are less than 500 micrometers across in one dimension where the film covers the holes. The film is translucent to x-rays. The present invention also includes an apparatus for preparing samples for measurement by x-ray fluorescence spectrometry. The apparatus comprises a plate having one or more holes passing through the plate. The holes are covered on one side of the plate by a detachable cover forming a water-tight seal against the plate. The cover is substantially free of the elements osmium, yttrium, iridium, phosphorus, zirconium, platinum, gold, niobium, mercury, thallium, molybdenum, sulfur, lead, bismuth, technetium, ruthenium, chlorine, rhodium, palladium, argon, silver, and thorium. The holes are less than about 500 micrometers across in one dimension where the cover covers the holes. The present invention also includes a method for preparing samples for measurement by x-ray fluorescence spectrometry. The method comprises providing a solution of with less than 10 micromolar solute and a volume of between about 2 microliters and about 2 milliliters. The solution is concentrated and analyzed using x-ray fluorescence spectrometry.

RELATED APPLICATIONS

This application is related to and claims the priority of U.S.Provisional Patent Application 60/965,052 entitled “Well Plate,” whichwas filed on Aug. 16, 2007, which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the preparation of samples forspectroscopic analysis.

BACKGROUND OF THE INVENTION

X-ray fluorescence (XRF) spectrometry is a powerful spectroscopictechnique that has been used to determine the elements that are presentin a chemical sample, and to determine the quantity of those elements inthe sample. The underlying physical principle of the method is that whenan atom of a particular element is irradiated with X-ray radiation, theatom ejects a core electron such as a K shell electron. The resultingatom is then in an excited state, and it can return to the ground stateby replacing the ejected electron with an electron from a higher energyorbital. This is accompanied by the emission of a photon. The energy ofthe emitted photons is equal to the difference in the energies of thetwo orbitals. Each element has a characteristic set of orbital energiesand therefore, a characteristic X-ray fluorescence (XRF) spectrum.

An XRF spectrometer is an apparatus capable of irradiating a sample withan X-ray beam, detecting the X-ray fluorescence from the sample, andusing the X-ray fluorescence to determine which elements are present inthe sample and measuring the quantity of these elements. A typical,commercially available energy dispersive X-ray fluorescence spectrometeris the EDAX Eagle XPL energy dispersive X-ray fluorescence spectrometer,equipped with a microfocus X-ray tube, lithium drifted siliconsolid-state detector, processing electronics, and vendor suppliedoperating software, available from the EDAX division of Ametek, 91 McKeeDrive Mahwah, N.J. 07430. An example of a wavelength dispersive X-rayfluorescence spectrometer is the ZSX Primus, available from RigakuAmericas, 9009 New Trails Drive, The Woodlands, Tex. 77381. Inprinciple, any element may be detected and quantified with XRF.

Typical protein-drug assays are performed with nanomolar to micromolarprotein concentrations and drug concentrations. The proteinconcentration and the drug concentration need not be the same. Theexisting art for the analysis of dried samples by x-ray fluorescence canonly measure dried samples which are deposited from one microliter orless of solutions having minimum concentrations of about 10 micromolar(see Thomasin C. Miller, Christopher M. Sparks, George J. Havrilla,Meredith R. Beebe, Semiconductor applications of nanoliter dropletmethodology with total reflection X-ray fluorescence analysis,Spectrochimica Acta Part B 59 (2004) 1117-1124, incorporated herein byreference). This sample preparation method is insufficient for theanalysis of proteins and protein-drug complexes, where biologicallyrelevant concentrations of the solution from which the sample isdeposited must be less than 10 micromolar and preferably less than 100nanomolar.

The existing state of the art is insufficient for analyzing dilutesolutions, especially dilute solutions of proteins having concentrationsof less than about 10 micromolar.

There remains a need for simpler methods for preparing samples formeasurement using x-ray fluorescence spectrometry. The present inventionis designed to address that need.

SUMMARY OF THE PRESENT INVENTION

One aspect of the present invention includes an apparatus for preparingsamples for measurement by x-ray fluorescence spectrometry. Theapparatus comprises a plate having one or more holes passing through theplate. The holes are covered by a film on one side of the plate. Theholes are less than 500 micrometers across in one dimension where thefilm covers the holes. The film is translucent to x-rays.

Another aspect of the present invention comprises an apparatus forpreparing samples for measurement by x-ray fluorescence spectrometry.The apparatus comprises a plate having one or more holes passing throughthe plate. The holes are covered on one side of the plate by adetachable cover forming a water-tight seal against the plate. The coveris substantially free of the elements osmium, yttrium, iridium,phosphorus, zirconium, platinum, gold, niobium, mercury, thallium,molybdenum, sulfur, lead, bismuth, technetium, ruthenium, chlorine,rhodium, palladium, argon, silver, and thorium. The holes are less thanabout 500 micrometers across in one dimension where the cover covers theholes.

Still another aspect of the present invention includes a method forpreparing samples for measurement by x-ray fluorescence spectrometry.The method comprises providing a solution with less than 10 micromolarsolute and a volume of between about 2 microliters and about 2milliliters. The solution is concentrated and analyzed using x-rayfluorescence spectrometry.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic depiction of the present invention.

FIG. 2 shows a cutaway view of Plate 4 showing examples of the shapes ofHole(s) 8.

FIG. 3 shows protein samples prepared using Apparatus 2 and by pipette.

FIG. 4 shows x-ray fluorescence spectra of protein samples preparedusing Apparatus 2 and by pipette.

FIG. 5 shows a comparison of the cross-sectional thickness profile ofprotein samples prepared using Apparatus 2 and by pipette.

DETAILED DESCRIPTION

Briefly, the present invention includes an apparatus for preparingsamples for measurement by x-ray fluorescence spectrometry. Theapparatus comprises a plate having one or more holes passing through theplate. The holes are covered by a film on one side of the plate. Theholes are less than 500 micrometers across in one dimension where thefilm covers the holes. The film is translucent to x-rays. The presentinvention also includes an apparatus for preparing samples formeasurement by x-ray fluorescence spectrometry. The apparatus comprisesa plate having one or more holes passing through the plate. The holesare covered on one side of the plate by a detachable cover forming awater-tight seal against the plate. The cover is substantially free ofthe elements osmium, yttrium, iridium, phosphorus, zirconium, platinum,gold, niobium, mercury, thallium, molybdenum, sulfur, lead, bismuth,technetium, ruthenium, chlorine, rhodium, palladium, argon, silver, andthorium. The holes are less than about 500 micrometers across in onedimension where the cover covers the holes. The present invention alsoincludes a method for preparing samples for measurement by x-rayfluorescence spectrometry. The method comprises providing a solutionwith less than 10 micromolar solute and a volume of between about 2microliters and about 2 milliliters. The solution is concentrated andanalyzed using x-ray fluorescence spectrometry.

The present invention comprises Apparatus 2, which includes Plate 4 andFilm 6. Film 6 covers one face of Plate 4. Plate 4 has one or more Holes8. Holes 8 pass through Plate 4 from the face of Plate 4 which iscovered by Film 6 to the opposite face of Plate 4. Film 6 may be bondedto Plate 4.

Apparatus 2 should not leak solvent at the junction of Film 6 and Plate4. Preferably, the junction of Film 6 and Plate 4 maintains awater-tight seal when Apparatus 2 is subjected to either temperatures inexcess of about 100° F., or acceleration in excess of about 20 times theearth's gravitational constant at sea level, or atmospheric pressureless than about 760 torr.

The dimensions of Plate 4 are more preferably 127.76 mm±0.25 mm by 85.48mm±0.25 mm, when measured from a position within 12.7 mm (0.5000 inches)of the outside corners of Plate 4. If Plate 4 is substantiallyrectangular or if Plate 4 has a flange on the bottom, then Plate 4 orits flange preferably has a corner radius to the outside of 3.18 mm±1.6mm. If Plate 4 is substantially rectangular with has one or morechamfers or beveled corners, then the corner radii of any non-beveledcorners are preferably 3.18 mm±1.6 mm to the outside. If Apparatus 2comprises 96 Hole(s) 8, then Hole(s) 8 are preferably arranged as eightrows by twelve columns. In this case, the distance between at least oneoutside edge of Plate 4 and the center of the first column of Hole(s) 8is preferably 14.38 mm±4 mm. The distance between each column of Hole(s)8 is preferably 9 mm±4 mm. The distance between at least one outsideedge of Plate 4 and the center of the first row of Hole(s) 8 ispreferably 11.24 mm±4 mm. The distance between each row of Hole(s) 8 ispreferably 9 mm±4 mm. At least one of Hole(s) 8 is preferably marked ina distinguishing manner, and more preferably each row is marked in adistinguishing manner and each column is marked in a distinguishingmanner. If Apparatus 2 comprises 384 Hole(s) 8, then Hole(s) 8 arepreferably arranged as sixteen rows by twenty-four columns. In thiscase, the distance between at least one outside edge of Plate 4 and thecenter of the first column of Hole(s) 8 is preferably 12.13 mm±4 mm. Thedistance between each column of Hole(s) 8 is preferably 4.5 mm±2 mm. Thedistance between at least one outside edge of Plate 4 and the center ofthe first row of Hole(s) 8 is preferably 8.99 mm±4 mm. The distancebetween each row of Hole(s) 8 is preferably 4.5 mm±2 mm. At least one ofHole(s) 8 is preferably marked in a distinguishing manner, and morepreferably each row is marked in a distinguishing manner and each columnis marked in a distinguishing manner. If Apparatus 2 comprises 1536Hole(s) 8, then Hole(s) 8 are preferably arranged as thirty-two rows byforty-eight columns. In this case, the distance between at least oneoutside edge of Plate 4 and the center of the first column of Hole(s) 8is preferably 11.005 mm±4 mm. The distance between each column ofHole(s) 8 is preferably 2.25 mm±1 mm. The distance between at least oneoutside edge of Plate 4 and the center of the first row of Hole(s) 8 ispreferably 7.865 mm±4 mm. The distance between each row of Hole(s) 8 ispreferably 2.25 mm±1 mm. At least one of Hole(s) 8 is preferably markedin a distinguishing manner, and more preferably each row is marked in adistinguishing manner and each column is marked in a distinguishingmanner. Plate 4 is preferably between 7 mm and 35 mm in height. Top Face12 and Bottom Face 10 are preferably substantially parallel to eachother. Plate 4 may comprise a flange on one or more sides of Plate 4. IfPlate 4 comprises a flange, then the height of the flange is preferablybetween 1 mm and 10 mm, and more preferably is 2.41 mm±0.38 mm or 6.10mm±0.38 mm or 7.62 mm±0.38 mm when measured from the bottom-restingplane of Plate 4 to the top of the flange. It is preferable that ifPlate 4 is substantially rectangular, with or without chamfers on anycorners, that the flanges on at least two of the sides of Plate 4 havethe same flange height when measured from the bottom-resting plane ofPlate 4 to the top of the flange. The flange preferably extends fromPlate 4 by at least 0.5 mm and preferably by 1.27 mm when measured atthe top of the flange. Any chamfers preferably do not have flanges. Aflange on Plate 4 may have interruptions or projections. Preferably, anyedge of any interruption or projection on a flange is a minimum of 47.8mm from the edges of Plate 4 which are substantially perpendicular tothe flange having the interruption(s) or projection(s).

Plate 4 is preferably composed of plastic, although the use of metal tocompose Plate 4 will make Plate 4 more durable. Plate 4 preferably hasdimensions of a length of 5.0299 inches (plus or minus 0.5 inches) by awidth of 3.3654 inches (plus or minus 0.5 inches). The height of Plate 4is preferably between 1 millimeter and 150 millimeters. Plate 4 mostpreferably conforms to ANSI/SBS 1-2004 and ANSI/SBS 2-2004. Plate 4 hasa Top Face 12 and a Bottom Face 10.

Plate 4 has one or more Holes 8, which pass through Bottom Face 10 ofPlate 4 and Top Face 12 of Plate 4. If there are ninety-six (96) Holes8, or three hundred and eighty-four (384) Holes 8, or one thousand fivehundred and thirty-six (1,536) Holes 8, then the positioning of theHoles 8 preferably conforms to ANSI/SBS 4-2004. Holes 8 pass throughPlate 4. On the Top Face 12, Hole 8 has a diameter of between 500micrometers and thirty (30) millimeters. On the Bottom Face 10, Hole(s)8 have a diameter of between 10 micrometers and two millimeters, andpreferably a diameter of between 50 micrometers and 500 micrometers, andmost preferably a diameter of between 50 micrometers and 350micrometers. Each Hole 8 preferably has a volume sufficient to hold atleast 10 microliters between the planes defined by Top Face 12 andBottom Face 10. Each Hole 8 may optionally be coated with silicone oranother coating that minimizes protein binding to the walls of Hole 8.Hole(s) 8 may be conical or truncated conical with the wide end of thecone at the Top Face 12 of Plate 4 and the narrow end of the cone at theBottom Face 10 of Plate 4. Hole(s) 8 may be cylindrical at the top andconical or truncated conical at the bottom. Hole(s) 8 may be cylindricalat the top and conical or truncated conical in the middle, andcylindrical at the bottom. Hole(s) 8 may be cylindrical at the top andconical or truncated conical in the middle, and conical at the bottomwith wide end of the bottom cone opening onto Bottom Face 10 of Plate 4;in this configuration, Hole(s) 8 are dumbbell shaped. Any shape whichallows liquid to flow through Hole(s) 8 is acceptable. The perimeter atthe widest part of Hole(s) 8 is preferably greater than the perimeter ofHole(s) 8 where Hole(s) 8 is covered by Film 6.

Hole(s) 8 should be chemically inert under the conditions of use.Preferably, Hole(s) 8 do not release contaminants that interfere withthe XRF measurements of Sample 18. More preferably, Holes(8) do notrelease more than 5 parts per billion sulfur or 5 parts per billionchlorine or five parts per billion phosphorus in any chemical form whenexposed to a solution comprising water, proteins, dimethylsulfoxide ordimethylformamide at 50° C. for 3 hours.

Hole(s) 8 are also preferably smooth so that Sample 18 does not becomeentrained in rough surfaces. Any surface features in Hole(s) 8preferably are less than 200 microns in the dimension normal to theinside face of Hole(s) 8. The inside face of Hole(s) 8 preferably has aroot mean squared (RMS) roughness of less than about 20 micrometers. AHole (8) having an internal depression of 200 micrometers functionedacceptably in the present invention. Hole (8) having a roughness ofapproximately 5 micrometers functioned acceptably in the presentinvention. To minimize the amount of Sample 18 that becomes adhered tothe side of Hole(s) 8, Hole(s) 8 should have an included angle of lessthan about 45 degrees, and more preferably less than about 42 degrees,and most preferably less than about 40 degrees. The entire Hole(s) 8 canhave these included angles, or just the portion of Hole(s) 8 where thecross sectional area is being reduced can be shaped to have thisincluded angle.

The line defining the interface between the solution, the solid surfaceof Hole(s) 8, and the atmosphere is called the contact line. Hole(s) 8are preferably shaped so that the length of the contact line diminishesfor at least a portion of the time in which the volume of the solutionis reduced. It is desirable that a portion of Sample 18 adhere to Film 6if Film 6 is detached; therefore, it is desirable that Hole(s) 8 bewider where it adjoins Film 6 than where Hole(s) 8 is at its narrowest.

The Hole(s) 8 should have a small cross sectional area at the bottom,i.e. the location where the Hole(s) 8 adjoins Film 6. The cross sectionof Hole(s) 8 at this location are preferably less than about 500micrometers across in one dimension, and are more preferably less than1000 micrometers across in the other dimension, so that the entire crosssectional area of Hole(s) 8 at the location where Hole(s) 8 adjoin Film6 is less than about 0.005 square centimeters.

Film 6 is located across the Bottom Face 10 of Plate 4. Film 6 may beheld against Bottom Face 10 of Plate 4 by an optional Adhesive 14.Alternatively, Film 6 may be held against Bottom Face 10 of Plate 4 byan optional Bottom Plate 16, in which case Film 6 is placed betweenBottom Plate 14 and Bottom Face 10 of Plate 4. A Gasket 20 may be placedbetween Film 6 and Bottom Plate 16. Film 6 may be bonded to Bottom Face10 of Plate 4 by heat or by a solvent. Film 6 may be permanentlyattached to Plate 4 or it may be removable. Film 6 is preferablysubstantially free of the at least one of the elements sulfur,phosphorus, or chlorine. Examples of suitable materials include Porvair#229302, Porvair #229112, Porvair #229058, Porvair #229304, and Porvair#229301, all of which are available from Porvair plc, Brampton House, 50Bergen Way, King's Lynn, Norfolk PE30 2JG, U.K.), aluminum foils(examples: Microseal ‘F’ Foil from Bio-Rad Laboratories, 1000 AlfredNobel Drive, Hercules, Calif. 94547). Other tapes that may be used forFilm 6 include: Super-Thin Polyester Surface-Protection Tape,Chemical-Resistant Surlyn Surface-Protection Tape, Abrasion-ResistantPolyurethane Surface-Protection Tape, Heat-Resistant Kapton Tape withSilicone Adhesive or with Acrylic Adhesive, UV-Resistant PolyethyleneSurface-Protection Tape, Clean-Release Polyethylene Surface-ProtectionTape, Low-Static Polyimide Tape, all available from McMaster-Carr, 6100Fulton Industrial Blvd., Atlanta, Ga. 30336-2852. Other materials whichmay be used for Film 6 include 0.02 micrometer thick, 99.99% pure goldfoil, available from Lebow Company, 5960 Mandarin Ave., Goleta, Calif.93117 U.S.A.; 0.07 micrometer thick, 99.95% pure aluminum foil,available from Lebow Company, 5960 Mandarin Ave., Goleta, Calif. 93117U.S.A.; 0.1 micrometer through 20 micrometer thick PARYLENE N®,available from Lebow Company, 5960 Mandarin Ave., Goleta, Calif. 93117U.S.A.; and 0.5 micrometer thick to 25.0 micrometer thick, 99.99% puretitanium foil, available from Lebow Company, 5960 Mandarin Ave., Goleta,Calif. 93117 U.S.A.; and 4 micrometer thick to 8 micrometer thickpolypropylene, available from Lebow Company, 5960 Mandarin Ave., Goleta,Calif. 93117 U.S.A. Other substrates that are conveniently used are AP1,AP3, ProLINE Series 10, ProLINE Series 20, DuraBeryllium substrates fromMoxtek, 452 West 1260 North, Orem, Utah 84057. Other materials which maybe used for Film 6 are Ultralene®, mylar, polycarbonate, prolene, andkapton, available from SPEX CertiPrep Ltd, 2 Dalston Gardens, Stanmore,Middlesex HA7 1 BQ, ENGLAND. Other materials that may be convenientlyused are Hostaphan®, polyester, and Etnom® available from ChemplexIndustries, Inc., 2820 SW 42nd Avenue, Palm City, Fla. 34990-5573 USA.Another material that may be conveniently used is Zone Free Film PartZAF-PE-50, available from Excel Scientific, 18350 George Blvd,Victorville, Calif., 92394. This list is not exhaustive, and othermaterials may be used for Film 6. Film 6 may be made of fluorocarbons.Film 6 is also preferably substantially free of elements which haveX-Ray Fluorescence emission peaks having energies of between 1.9 KeV and3 KeV, because these peaks tend to interfere with the signals of mostinterest for the application of Apparatus 2 to biochemical andbiological applications. Elements which have X-Ray Fluorescence emissionpeaks having energies of between 1.9 KeV and 3 KeV are: osmium, yttrium,iridium, phosphorus, zirconium, platinum, gold, niobium, mercury,thallium, molybdenum, sulfur, lead, bismuth, technetium, ruthenium,chlorine, rhodium, palladium, argon, silver, and thorium. If Apparatus 2is used with an x-ray fluorescence spectrometer which uses an x-raydetector which comprises silicon, then Film 6 is also preferably free ofelements which have X-Ray Fluorescence escape peaks (i.e. x-rayfluorescence emission peaks minus 1.74 KeV) having energies of between1.9 KeV and 3 KeV, because these escape peaks tend to interfere with thesignals of most interest for the application of Apparatus 2 tobiochemical and biological applications. Elements which have X-RayFluorescence escape peaks having energies of between 1.9 KeV and 3 KeVare: calcium, tellurium, iodine, scandium, xenon, cesium, barium,titanium, and lanthanum. “Substantially free” is defined herein as beingless than about 4% by weight. Film 6 may have additional chemicalelements, which may be used for measuring the thickness of Sample 18. Ifwavelength dispersive x-ray fluorescence is used, then the elementalpurity of Film 6 is not as important; in this case, the film should besubstantially free of the element or elements which are being used toquantify the sample. Film 6 may be treated to increase protein adhesion;a non-inclusive list of treatments includes treating Film 6 with oxygenor nitrogen plasma or with poly-lysine.

If Film 6 is bonded to Plate 4, then the Film 6 should deform withSample 18. Film 6 deforms from the plane defined by the perimeter ofHole(s) 8 where Hole(s) 8 adjoins Film 6, by a minimum distance of 100nanometers.

If Film 6 is detachable from Plate 4, then the Plate 4 deforms as aresult of loading during normal use. Plate 4 is deformed from the planeFilm 6 by a minimum distance of 10 micrometers.

If Film 6 is not detachable from Bottom Face 10 of Plate 4, then Film 6is preferably translucent to x-rays having energies of 2.3 KeV. In thiscase, translucent is defined as allowing at least 5% of the x-rays whichare normal to Film 6 and which have an energy of 2.3 KeV to pass throughFilm 6, when Film 6 is placed in a plane which is perpendicular to aline defined by Sample 18 and an x-ray detector. The materialcompositions and thicknesses which may be used for Film 6 (if Film 6 isnot detachable from Bottom Face 10 of Plate 4) may be measured orcalculated using x-ray attenuation parameters for various materials. Anon-exhaustive list of materials which are suitable for Film 6 (if Film6 is not detachable from Bottom Face 10 of Plate 4) includespolypropylene which is less than about 195 micrometers thick, andpolycarbonate which is less than about 105 micrometers thick.

Apparatus 2 is used with an x-ray fluorescence spectrometer. Apparatus 2is preferably used by placing a solution of Sample 18 in one or moreHole(s) 8. At least a portion of the solvent containing Sample 18 isthen evaporated. Preferably at least 80% of the solvent of Sample 18 isevaporated; more preferably, substantially all of the solvent of Sample18 is evaporated. The solvent is preferably evaporated using elevatedtemperatures that are above about 22° C. or reduced atmospheric pressurethat is below about 760 torr. Apparatus 2 is preferably placed in avacuum centrifuge, such as a Savant Speed Vac Plus SC 250DDA or a ThermoSavant SPD 1010 SpeedVac®. Solvent is preferably removed from Sample 18while Apparatus 2 is centrifuged, and the Sample 18 is concentrated bybeing collected at the portion of Hole 8 which passes through BottomFace 10 of Plate 4 and onto Film 6. The advantage of the Apparatus 2 isthat the sample is concentrated into a small area. If Film 6 isdetachable and Film 6 is thick enough that it is not translucent(translucent is defined as allowing at least 5% of the x-rays which arenormal to Film 6 and which have an energy of 2,300 eV to pass throughFilm 6, when Film 6 is placed in a plane which is perpendicular to aline defined by Sample 18 and an x-ray detector), then Film 6 is thendetached from Plate 4 and measured in an X-ray Fluorescence Spectrometer(for example, an EDAX Eagle III μprobe, available from EDAX, 91 McKeeDrive Mahwah, N.J. 07430; or a MicroXR VXR Microbeam XRF System,available from Thermo Fisher Scientific, Inc., 81 Wyman Street, Waltham,Mass. 02454). In this case, Film 6 is oriented in the X-ray fluorescencespectrometer so that Sample 18 is between Film 6 and the x-ray detectorof the x-ray fluorescence spectrometer. Film 6 is preferably held in aFrame 20 to hold Film 6 flat. If Film 6 is translucent to x-rays asdefined above, then Sample 18 may be measured without detaching Film 6from Plate 4. If Film 6 is translucent to x-rays, then Sample 18 may bemeasured using an x-ray fluorescence spectrometer with Film 6 orientedbetween Sample 18 and the x-ray detector, whether or not Film 6 isdetached from Plate 4. The advantage of not detaching Film 6 from Plate4 is that Plate 4 functions as a frame, and therefore no additionalFrame 20 is necessary. The advantage of detaching Film 6 is that Sample18 may be measured with no attenuation from Film 6. Apparatus 2 ispreferably used with an x-ray fluorescence spectrometer whose beam sizeis matched to the size of Sample 18 after the solvent has beenevaporated; in this case a matched beam size is defined as the area ofthe x-ray excitation beam which contains at least 80% of the x-ray fluxwhich is incident on Sample 18 is within a factor of 100 of the area ofSample 18. The x-ray excitation source may be focused, such as a bymeans of a polycapillary focusing optics offered by X-Ray OpticalSystems, Inc., 15 Tech Valley Drive, East Greenbush, N.Y. 12061. Anx-ray fluorescence spectrometer equipped with a collimator or anothertype of focusing optic on the x-ray excitation source is alsoacceptable.

The advantage of using Apparatus 2 for preparing samples for x-rayfluorescence spectrometry measurements is that the measurement limitsare better when using Apparatus 2 versus simply pipetting or printing asample on a film as described in Miller T C et al. “Semiconductorapplications of nanoliter droplet methodology with total reflectionx-ray fluorescence analysis.” Spectrochimica Acta B. 2004, 59:1117-1124;or Miller T C and G J Havrilla. “Nanodroplets: a new method for driedspot preparation and analysis” X-Ray Spect 2004, 33:101-106, forexample. The second advantage of using Apparatus 2 is that manybiological samples are prepared as dilute solutions, for example asdilute solutions of proteins. Apparatus 2 allows relatively largevolumes of a solution of sample to be prepared, and the sample to beconcentrated into a dried spot of sample with optimum properties.Volumes of solution are preferably between 2 microliters to 2milliliters, and more preferably are between 10 microliters and 250microliters. In contrast, existing sample preparation methods requirehighly concentrated sample solutions in order to deposit a sufficientamount of sample for measurement. Also in contrast, existing samplepreparation methods require a large amount of sample to prime theirprinter head or fill their reservoirs, which wastes valuable sample. Forexample, assume one desires to measure approximately 30 picograms ofsulfur in a sample. Using standard deposition methods, which might use10 nanoliters of solution, the concentration of sulfur in the initialsolution would have to be 100 micromolar. Using Apparatus 2, 100microliters of solvent may be used, resulting in an initial sampleconcentration of 10 nanomolar. For biology, biochemistry, and drugdevelopment, it is important to be able to work with solutions whichhave nanomolar concentration, versus solutions with tens or hundreds ofmicromolar concentrations. If Apparatus 2 is used with a protein or anucleic acid, the mass of the sulfur or phosphorus or a combination ofsulfur and phosphorus in the protein or nucleic acid is preferablybetween 50 femtograms and 1 microgram, and most preferably the mass ofthe phosphorus or sulfur or combinations thereof in Sample 18 is between100 picograms and 100 nanograms.

Sample 18 is deposited in a shape which is defined by the shape of Hole8 as it penetrates the Bottom Face 10 of Plate 4. The most efficientshape for Sample 18 is one which is similar to the size of the x-rayexcitation beam of the x-ray fluorescence spectrometer. Usually, bothSample 18 and the x-ray excitation beam are roughly circular in outline.Similarity, in this context, means that the area of Sample 18 is withina factor of 100 times greater or smaller than the area of the x-rayexcitation beam as it illuminates Sample 18 (for example, the diameterof Sample 18 is within a factor of 10 of the x-ray excitation beam as itilluminates Sample 18). Preferably, Sample 18 is within a factor of 25times bigger or smaller than the area of the x-ray excitation beam as itilluminates Sample 18 (for example, the diameter of Sample 18 is withina factor of 5 of the x-ray excitation beam as it illuminates Sample 18).If the area of x-ray excitation beam as it illuminates Sample 18 issignificantly greater than the area of Sample 18, then x-ray photons arewasted. If the area of x-ray excitation beam as it illuminates Sample 18significantly smaller than the area of Sample 18, then the measurementtime will be unnecessarily long or else a portion of Sample 18 will bewasted by its not being measured.

Sample 18 typically comprises an aqueous sample of a protein or nucleicacid, which has been optionally modified by addition of an inhibitor,co-factor, metal, protein, sugar, or other chemical. Sample 18 may beconveniently measured using an X-ray fluorescence instrument. This x-rayfluorescence instrument preferably comprises at least one of thefollowing: a monocapillary focusing optic, polycapillary focusing optic,a collimator, a microfocus X-ray tube, a synchrotron X-ray source, alinear accelerator X-ray source, a rhodium X-ray tube, a molybdenumX-ray tube, a chromium X-ray tube, a silver X-ray tube, a palladiumX-ray tube, a monochromatic X-ray source, a polychromatic X-ray source,a polarized X-ray source, a confocal X-ray fluorescence spectrometerfocusing arrangement, a PIN diode detector, a semiconductor X-raydetector, a germanium or doped germanium X-ray detector, a silicon ordoped silicon X-ray detector, a wavelength dispersive X-ray fluorescencespectrometer, an energy dispersive X-ray fluorescence spectrometer,total reflectance X-ray fluorescence spectrometer, and the like.

If Film 6 contains additional chemical elements which are measurablewith the x-ray fluorescence spectrometer being used with Apparatus 2,then these elements may be used to measure the thickness of Sample 18.For example, if Film 6 contains silicon, then the attenuation of thesilicon x-ray signal by Sample 18 will allow the thickness of Sample 18to be estimated. FIG. 5 shows the relative thicknesses of a proteinsample deposited using Apparatus 2 and a protein sample deposited usinga pipette. The protein sample deposited using Apparatus 2 issignificantly more compact and lacks the ring shape as compared withsamples deposited by pipette which display alternating rings ofdeposited sample and rings that are depleted in sample. FIG. 3 shows aphotograph of a sample deposited by pipette. FIG. 3 also shows aphotograph of a sample deposited using Apparatus 2. The sample depositedusing a pipette is significantly larger and has more rings than thesample deposited using Apparatus 2. The more compact sample depositedusing Apparatus 2 provides a more intense signal in the x-rayfluorescence spectrum, as shown in FIG. 4. Thickness corrections areperformed by measuring the signal from Film 6 (e.g. a silicon signal) ina region where there is no Sample 18, and measuring the same elementalsignal from a region of Film 6 which is covered by Sample 18. Thedifference in the x-ray signal from the film may be related to thethickness of Sample 18 by calculating the thickness of Sample 18required to attenuate the x-ray signal by the measured amount.

Apparatus 2 may be used with proteins. Many proteins require a buffer tomaintain the pH within a particular range (e.g. a pH buffer), or tomaintain the redox state of a chemical (e.g. a redox buffer), or tomaintain an ionic strength (e.g. an isotonic buffer). Many bufferscontain elements which might interfere with the measurement of thechemical. The buffer should preferably be free of at least one chemicalelement having an atomic number of greater than four, where thatchemical element is present in Sample 18. The buffer should morepreferably be free of at least one chemical element having an atomicnumber of greater than eight, where that chemical element is present inthe Sample 18. The buffer should preferably be free of at least one ofthe following chemicals or functional groups: dimethylsulfoxide, thiols,sulfate anion, sulfonate anions, chloride anion, bromide anion, fluorideanion, iodide anion, perchlorate anion, phosphate anion, and phosphonateanions. The buffer preferably comprises one or more of the followingchemical or functional groups: amine, imine, nitrate anion, nitriteanion, ammonium cation, acetate anion, carboxylate anion, carbonateanion, and iminium cation; these chemicals offer the correct bufferingproperties with minimal x-ray fluorescence interference. Mostpreferably, the buffer comprises an ammonium nitrate salt such astris(ethanol)amine nitrate, also known as tris nitrate.

The proteins to be used with the present invention are preferablypurified to remove chemicals, including pH buffers and redox buffers andisotonic buffers, that are not chemically bound or which are looselybound to the protein. This purification also removes salts thatcontribute to poor sample quality. Loosely bound is defined as meaninghaving a binding affinity that is weaker than about ten millimolar. Thisdesalting step may be conducted conveniently using gel filtrationchromatography or size exclusion chromatography, such a Quick SpinProtein Column using Sephadex G-25, available from Roche AppliedScience, PO Box 50414, Indianapolis, Ind., 46250. This process isamenable to multiplexing using a well plate format, such as a 96-well,384-well, or 1536-well plate format. Separations systems such as Zeba96-well plates available from Pierce Biotechnology Inc., PO Box 117,Rockford, Ill., 61105, are particularly convenient. The advantage ofremoving the buffers and other loosely bound chemicals is that theirremoval minimizes interfering elements which might attenuate the desiredX-ray fluorescence signal or add spurious x-ray fluorescence signals, orwhich create non-fluorescent x-ray signals through diffraction and othersimilar processes.

Both single-plexed or multiplexed gel filtration chromatography processmay be expedited using a centrifuge, such as a the IEC CL40 availablefrom Thermo Fisher Scientific, product #11210923, 450 Fortune Blvd,Milford, Mass., 01757; or a vacuum manifold, such as a Vacuum apparatussuch as the MultiScreen Vacuum manifold with Direct Stack fromMillipore, 290 Concord Road, Billerica, Mass. 01821, attached to astandard vacuum pump (for example, Millipore, Catalog # WP61 115 60)also available from Millipore. Separations are preferably carried out inless than 300 seconds of centrifugation, and are more preferably carriedout in less than 30 seconds of centrifugation. An alternative method ofseparation is ultrafiltration, such as might be performed using aCentricon YM-3 centrifuged for 3 hours at 7000 g.

Desalting by size exclusion columns may be conveniently used withApparatus 2, by using a desalting well plate having the same number ofwells and locations of wells as the number and location(s) of Hole(s) 8in Apparatus 2. The desalting plate is placed on top of Apparatus 2, anda solution of Sample 2 is placed in one or more of the wells of thedesalting plate. The stacked desalting plate and Apparatus 2 arecentrifuged until solution containing Sample 18 passes through thedesalting plate and into Hole(s) 8 of Apparatus 2. Apparatus 2 is thenplaced in a vacuum centrifuge as described above. Alternatively, thesolution of Sample 18 may be desalted and transferred to Apparatus 2,for example by pipette.

If Sample 18 comprises a protein, it should preferably be present at aconcentration of less than 10 micromolar and more preferably less than100 nanomolar. In addition to proteins, other biological molecules whichmay be used include nucleic acids, polysaccharides, peptides, and otherbiologically derived molecules; like proteins, these are preferablypresent in solution at concentrations less than 10 micromolar and morepreferably less than 100 nanomolar.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

The embodiment(s) were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. An apparatus for preparing one or more samplesfor measurement by X-ray fluorescence spectrometry, the apparatuscomprising: a plate having at least one hole passing through an entirethickness of the plate, wherein the hole is configured to receive atleast one sample having a volume of between 2 microliters and 2milliliter and to be prepared for measurement by X-ray fluorescencespectrometry; and a film covering one side of the hole such that thefilm is configured to receive the sample deposited into the hole,wherein the film allows less than 5% of the x-rays that are normal tothe firm and have an energy of 2,300 eV to pass therethrough, and thefirm is removable from the plate for analysis of the sample depositedtherein by X-ray fluorescence spectrometry.
 2. The apparatus of claim 1,wherein the plate is chemically inert under conditions of use.
 3. Theapparatus of claim 1, wherein the hole is shaped so that a length of acontact line diminishes for at least a portion of a time in which thevolume of a solution in the hole is reduced, and wherein the contactline is defined as an interface between the solution and a solid surfaceof the hole.
 4. The apparatus of claim 1, wherein the hole is configuredto receive the sample having a volume of between 10 microliters and 250microliters to be prepared for measurement by X-ray fluorescencespectrometry.
 5. The apparatus of claim 1, wherein a diameter of thehole at the location where the hole adjoins the film is within a factorof 100 times greater or 100 times smaller than an area of an X-rayexcitation beam as it illuminates the sample.
 6. The apparatus of claim1, further comprising an x-ray fluorescence spectrometer capable ofproducing an x-ray excitation beam, wherein the x-ray excitation beamhas a beam cross sectional area containing at least 80% of the x-rayflux of the x-ray excitation beam at a location where the x-rayexcitation beam is incident on the sample, and wherein the beam crosssectional area at the location is within a factor of 100 of an area ofthe sample.
 7. An apparatus for measurement by X-ray fluorescencespectrometry, the apparatus comprising: a plate having at least one holepassing through an entire thickness of the plate, wherein the at leastone hole comprises at least one sample; a film covering the holeoriented normal to the film, wherein the film is removable from theplate and a thickness of the film is at least 5 times smaller than athickness of the plate, and wherein the film allows less than 5% of thex-rays that are normal to the firm and have an energy of 2,300 eV topass therethrough; and an x-ray fluorescence spectrometer being capableof producing an x-ray excitation beam, the x-ray excitation beam havinga size that is matched to a size of the sample.
 8. The apparatus ofclaim 7, wherein the x-ray excitation beam has a beam cross sectionalarea containing at least 80% of the x-ray flux of the x-ray excitationbeam at a location where the x-ray excitation beam is incident on thesample, and wherein the beam cross sectional area at the location iswithin a factor of 100 of an area of the sample.
 9. The apparatus ofclaim 8, wherein the area of the x-ray excitation beam, as the x-rayexcitation beam illuminates the sample, is within a factor of 100 timesgreater or smaller than the diameter of the sample.
 10. The apparatus ofclaim 7, wherein the hole is configured to receive a volume of asolution between 2 microliters and 2 milliliters to be prepared formeasurement by X-ray fluorescence.
 11. The apparatus of claim 7, whereinthe hole is configured to receive a volume of a solution between 10microliters and 250 microliters to be prepared for measurement by X-rayfluorescence.
 12. The apparatus of claim 7, wherein a diameter of thehole at a location where the hole adjoins is covered by the film iswithin a factor of 100 times greater or 100 times smaller than an areaof an X-ray excitation beam as the x-ray excitation beam illuminates thesample.
 13. A system for preparing one or more samples for measurementby X-ray fluorescence spectrometry, the apparatus comprising: a platehaving at least one hole passing through the plate, wherein the hole isconfigured to receive a sample having a volume of between 2 microlitersand 2 milliliters and to be prepared for measurement by X-rayfluorescence spectrometry; a film covering the hole and being configuredto receive at least a portion of the sample deposited into the hole,wherein the film allows less than 5% of the x-rays that are normal tothe firm and have an energy of 2,300 eV to pass therethrough; and anx-ray fluorescence spectrometer capable of producing an x-ray excitationbeam, wherein the x-ray excitation beam has a beam cross sectional areacontaining at least 80% of the x-ray flux of the x-ray excitation beamat a location where the x-ray excitation beam is incident on the sample,and wherein the beam cross sectional area at the location is within afactor of 100 of an area of the sample.